The overall aim of this proposal is to develop an automated digital imaging system for simultaneously detecting hybridization of multiple probes to gridded arrays of DNA on solid membrane supports. Combinatorial fluorescence detection will be used to increase throughput to rates exceeding those of autoradiographic detection, at equivalent sensitivity levels. The proposed imaging method is based on a low-power epifluorescence microscope fitted with narrowband optical filters that permit high contrast discrimination between five spectrally distinguishable fluors. Used in boolean combination, these permit simultaneous use of up to 31 probes. The detector will be a cooled CCD camera. The readout rate limitations of the CCD will be circumvented by storing a large number of microscope fields on the chip, which is only read out when full. The hybridization array is imaged using a serial- parallel approach, in which successive portions of the membrane are presented to the camera using an automated x,y stage. A second, smaller automated stage moving synchronously with the first is used to move the CCD. All stage-scanning operations and optical filter changes will be under computer control, using scripts written for the device drivers embedded in a commercial software package (BDS Image). The computer will download the CCD data, reconstruct the array from corresponding serial images, and apply mask logic to decode the boolean intersections that comprise the spectral signature of each combinatorially-labelled probe. Sensitivities on the order of 10(6) to 10(8) fluor molecules / spot are anticipated, with data throughput rates on the order of a few minutes up to a couple of hours per hybridization membrane. These performance figures make the instrument suitable for high speed screening of YAC DNA libraries. The method could also be applied to reading Southern blots and sequencing gels.